(A, A') The equatorial actin ring (white arrowheads) in early elongating cells (A, 20.5 hpf) is relocated to the anterior pole (yellow arrowheads) after 70 min blebbistatin treatment (A'). (B, B') The anterior relocation of the actin ring and inhibition of cell elongation after 60-min blebbistatin treatment (B) is reversed by a 60 min wash (B'). (C) At 23 hpf, the elongated notochord cell has a broad equatorial actin ring (white arrowhead) that is associated with a prominent constriction (red arrowhead). After 45-min blebbistatin treatment, the ring is shifted to the anterior pole (yellow arrowhead), whereas the constriction is not. (D) Mean distance between the anterior lateral domain and the middle of the ring (red), and the mean ring width (black) over time. While the ring shifts toward the anterior pole, indicated by the decrease of the anterior basal domain, its width stays relatively constant (shaded in gray). After the ring reaches the anterior lateral domain, the width decreases. n = 5; error bars = SEM. (E, E') Kymograph of the shifting actin ring based on Video 2. Individual filaments (arrows) from the anterior and posterior edge move towards the center of the ring (the equator, indicated by the dashed line), which itself is shifting anteriorly. After a certain time, the equatorial bound movement of filaments becomes parallel to the movement of the ring (arrowheads). A diagram of the different movements is shown in E'. (F) Angles of single filament movement at specific times with respect to the center of the ring. Red (positive values), movement from posterior towards the center; yellow (negative values), movement from anterior towards the center. n = 2–7; error bars = SEM. (G) Fluorescence recovery after photobleaching in cells expressing mCherry-hActin. The entire actin ring region was bleached. Recovery is significantly slower in blebbistatin-treated cells. Control, n = 4; blebbistatin, n = 7; p = 0.012. Solid lines indicate a single exponential fit for the control (red curve) with a turnover time of 90 ± 3 s, and a double exponential fit for the blebbistatin treatment (green curve), with a fast fraction with the same turnover as the control, and a slow fraction (f = 70% ± 4%) with a turnover time of 7.8 ± 0.6 min. (H) Time-lapse frames of Video 3 showing the anterior movement of the equatorial ring (1), its disappearance, and the emergence of a second ring (2) at the equator. Red arrowheads indicate the circumferential constriction. (I) The kymograph illustrates the close succession of the second ring to the first ring. The red dot indicates the time when the first ring disappears at the anterior lateral domain, and the second ring begins to appear. (J) Time-lapse frames from Video 4 showing the emergence of a second (2) and third (3) ring with long-term blebbistatin treatment. Anterior to the left in all panels. Scale bars, 10 μm.